ABSTRACT Long noncoding RNAs (lncRNAs) play essential roles in mammalian development by repressing transcription of genetically linked (adjacent) protein coding genes. The archetypal repressive lncRNA is Xist, which silences nearly all genes along the inactive X chromosome. Other lncRNAs, such as Kcnq1ot1 and Airn, silence adjacent genes in smaller genomic intervals, and may function through related mechanisms. Collectively, dysregulated gene expression caused by altered function of repressive lncRNAs can cause cancers and genetic disorders that affect roughly 1 in 1,000 live births. Methods to attenuate or potentiate the biological activity of such lncRNAs could provide new means to control gene expression in disease relevant regions of the genome. However, the mechanisms by which repressive lncRNAs silence their target genes remain poorly defined, and it is unclear how modulation of their silencing potential might be achieved. The long-term goals of our research are (i) to determine the molecular mechanisms by which lncRNAs regulate gene expression and (ii) to apply this knowledge to advance human health. To these ends, we recently developed a reductionist assay to study mechanisms of lncRNA-induced gene silencing. Using the assay, we discovered a novel function of the Xist lncRNA that we hypothesize is essential for its gene silencing ability. In parallel, we developed a novel method to quantify sequence content of lncRNAs that can be used to predict lncRNA function. This algorithm ? to our knowledge the first of its kind ? allowed rational design of Xist-like synthetic lncRNAs and uncovered a recurrent sequence signature shared between Xist and hundreds of other lncRNAs, including many known to have repressive activity. Lastly, through chemical probing of Xist's secondary structure we discovered a structural context for this sequence signature. These data support our central hypothesis, that lncRNAs with a shared sequence signature silence gene expression through a common mechanism. To test this hypothesis, we will: determine the physiological consequences of Xist's novel function and the mechanism by which it occurs (Aim 1), determine the sequence and structural signatures required for silencing by Xist and related lncRNAs (Aim 2), and determine the trans-acting factors required for silencing induced by Xist-like lncRNAs (Aim 3). We are uniquely suited to pursue these objectives given our expertise, the expertise of our collaborators, and the data described in our application. In the course of our work, we will develop computational and experimental methods that can be applied to predict and study function of any lncRNA. Our study will provide novel insights into the roles of lncRNAs in cellular physiology and disease and may suggest protein and structural dependencies of repressive lncRNAs that can be exploited to control transcription in regions of the human genome that are essential for normal health and development.